U.S. patent application number 12/287198 was filed with the patent office on 2010-04-08 for natural product derivatives with antimalarial activity.
This patent application is currently assigned to Radix Pharmaceuticals, Inc.. Invention is credited to Shuren Zhu.
Application Number | 20100087469 12/287198 |
Document ID | / |
Family ID | 42076264 |
Filed Date | 2010-04-08 |
United States Patent
Application |
20100087469 |
Kind Code |
A1 |
Zhu; Shuren |
April 8, 2010 |
Natural product derivatives with antimalarial activity
Abstract
The present invention provides new chemical compositions with
desirable biological activity and toxicity profiles for the
enhanced treatment of malaria.
Inventors: |
Zhu; Shuren; (Potomac,
MD) |
Correspondence
Address: |
Shuren Zhu
13303 Sunny Brooke Place
Potomac
MD
20854
US
|
Assignee: |
Radix Pharmaceuticals, Inc.
|
Family ID: |
42076264 |
Appl. No.: |
12/287198 |
Filed: |
October 8, 2008 |
Current U.S.
Class: |
514/279 |
Current CPC
Class: |
A61K 36/53 20130101;
Y02A 50/411 20180101; A61K 31/4745 20130101; Y02A 50/30
20180101 |
Class at
Publication: |
514/279 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was supported by Public Health Service Grant
Number: 1R43AI077109-01 for Radix Pharmaceuticals, Inc. The U.S.
Government has a paid-up license in this invention and the right in
limited circumstances to require the patent owner to license others
on reasonable terms as provided for by the terms of Grant Number:
1R43AI077109-01 awarded by Public Health Service.
Claims
1. A method for the treatment of malaria comprising administering
to a subject a therapeutically effective amount of a compound
having the formula (I): ##STR00005## or a compound having the
formula (II): ##STR00006##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] Not Applicable.
REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM
LISTING COMPACT DISC APPENDIX
[0003] Not Applicable.
FIELD OF INVENTION
[0004] The present invention relates to new chemical compositions
that are effective for the treatment of malaria.
BACKGROUND OF THE INVENTION
[0005] Malaria is the best known protozoal disease, caused by one
of four species of the sporazoa type--Plasmodium falciparum, P.
vivax, P. ovale, and P. malariae. It is one of the most common
infectious diseases in at least 100 tropical and subtropical
countries in Africa, Southeast Asia, and South America. According
to WHO, one out of every seventeen people alive today will die from
a disease transmitted by the bite of a mosquito. Worldwide, malaria
infects 300-600 million people and kills about three million in a
year. The increasing prevalence of multiple drug resistant strains
of Plasmodium falciparum in most malaria endemic areas has
significantly reduced the efficacy of current anti-malarial drugs
for prophylaxis and treatment of this disease. Although drug
resistance is a common problem in the treatment of most microbial
infections, malaria and many neoplasms, the impact is more acute
for malaria chemotherapy because of the limited number of
clinically useful anti-malarial drugs.
[0006] Only six prescription drugs are available in the US for
treating and/or preventing malaria: Atovaquone/proguanil,
Doxycycline, Mefloquine, Primaquine, Chloroquine phosphate, and
Hydroxychloroquine sulfate. All of them are discovered more than 40
years ago. Serious side effects are common. Primaquine is the only
available causal prophylactics. It has a low therapeutic index. The
use of chloroquine is limited because of the worldwide emergence of
drug-resistant strains of P. falciparum and P. vivax. Proguanil has
a short half-life and strains of P. falciparum resistant to
proguanil are common. The newest antimalarial drug Mefloquine was
developed in the late 1960s. It is initially reserved by WHO for
use in regions where drug resistance to chloroquine is a serious
problem. However, problems have arisen with mefloquine use. The
cure rate for mefloquine-sulfadoxine-pyrimethamine treatment of P.
falciparum in Southeast Asia fell from 96% in 1985 to as low as 50%
in 1990. Mefloquine can also produce adverse neurological and
psychiatric reactions. Artemisinin and its derivatives are
currently under development. However, these compounds cannot be
used during pregnancy since they have shown fetotoxicity in rodent
models. The current generations of artemisinins possess poor
efficacy of monotherapy. Artemisinins do not interfere with hepatic
stages of parasite development and therefore have no causal
prophylactic value.
[0007] Therefore, novel medicinal agents are urgently needed to
overcome the emergence of resistance and to control an
ever-increasing number of epidemics caused by the malaria
parasite.
[0008] For decades, natural products have been a wellspring of
drugs and drug leads. According to a recent survey, 61% of the 877
small-molecule new chemical entities introduced as drugs worldwide
during 1981-2002 can be traced to or were inspired by natural
products. These include natural products (6%), natural product
derivatives (27%), synthetic compounds with natural-product-derived
pharmacophores (5%), and synthetic compounds designed on the basis
of knowledge gained from a natural product (that is, a natural
product mimic; 23%). In certain therapeutic areas, the productivity
is higher: 78% of antibacterials and 74% of anticancer compounds
are natural products or have been derived from, or inspired by, a
natural product.
[0009] Ocimum sanctum, popularly known as Tulsi in Hindi and Holy
Basil in English, is ubiquitous in Hindu tradition. In traditional
Ayurvedic system of medicine, several medicinal properties have
been attributed to this plant. Essential oil of Tulsi has
antibacterial, antifungal and antiviral properties. It inhibits the
growth of E coli, B. anthracis, M. tuberculosis. Extracts from the
plant have been found to possess anti-diabetic,
antistress/adaptogenic, as well as antiallergic and immunomodulator
effects. A variety of biologically active compounds have been
isolated from the leaves, barks, roots and seeds of Ocimum sanctum.
Those compounds include but are not limited to the following:
eugenol, carvacrol, caryophyllene, ursolic acid, apigenin,
luteolin, orientin, molludistin, vicenin, cirsilineol,
cirsimaritin, isothymusin, etc. Trials have shown excellent
antimalarial activity of Ocimum sanctum. Ayurvedic preparations
containing Ocimum sanctum, Allium stivum, Piper nigram and Curcuma
longa has been shown to possess antimalarial activity against
Plasmodium vivax and Plasmodium falciparum. As far as its
antimalarial effect is concerned essential oil of Ocimum sanctum
has also been found to possess insecticidal and larvicidal
activities against mosquitoes.
[0010] An ethanol extract of the dried root barks of Ocimum sanctum
exhibited considerable in vitro antimalarial activity to warrant
fractionation. On the basis of the initial activity of crude
extracts, attention was focused on the bioactivity-guided
fractionation of the EtOH extract of the dried barks, which
resulted in the isolation of two new antimalarial natural products,
Formula A and Formula B. Their chemical structures are shown in
FIG. 1. Formula A, bearing a trans-diol moiety, was converted into
the corresponding dimethyl ether compound Formula I. Formula B,
bearing a cis-diol moiety, was converted into the corresponding
methylene acetal compound Formula II. The chemical structures of
the natural product derivatives, Formula I and Formula II, are
shown in FIG. 2. The synthesis route is shown in FIG. 3.
[0011] Two Plasmodium falciparum malaria parasite clones, W2
(chloroquine resistant) and D6 (chloroquine sensitive), were
utilized for in vitro efficacy testing. All new compounds were also
tested for toxicity against human adult liver epithelial cells
(THLE-3). Natural products Formula A and Formula B showed modest
inhibitory activity against both chloroquine sensitive malaria
strain (D-6) and chloroquine resistant malaria strain (W-2). The
two derivative compounds Formula I and Formula II showed equally
potent inhibitory activity against both chloroquine sensitive
malaria strain (D-6) and chloroquine resistant malaria strain
(W-2). The IC.sub.50' s (concentration of compound that affords 50%
of inhibition) were superior to the positive controls, chloroquine
and mefloquine. Noticeably, these compounds also showed much
reduced toxicity.
[0012] In mice models, both Formula I and Formula II have shown
excellent blood schizonticidal activity and oral prophylactic
activity.
[0013] The present invention relates to new, more active and less
toxic natural product derivatives for the treatment of malaria.
SUMMARY OF THE INVENTION
[0014] The present invention provides new chemical compositions and
methods of isolation and synthesis and using as antimalarial agents
thereof. The present invention relates to improvements in the
chemotherapy of malaria through isolation and chemical synthesis of
new compounds with desirable biological activity and toxicity
profiles for enhanced treatment.
[0015] Accordingly, this invention provides new chemical compound
Formula I, whose chemical structure is shown in FIG. 2.
##STR00001##
[0016] The invention also provides new chemical compound Formula
II, whose chemical structure is shown in FIG. 2.
##STR00002##
[0017] Two natural product compounds were isolated and purified
from the plant Ocimum sanctum and their derivatives were
synthesized. In vitro and in vivo antimalarial activities were
evaluated. Natural products Formula A and Formula B showed modest
inhibitory activity against both chloroquine sensitive and
chloroquine resistant malaria strains. The two natural product
derivatives Formula I and Formula II showed equally effective and
potent inhibitory activity against both chloroquine sensitive and
chloroquine resistant malaria strains. It was observed that these
derivative compounds possessed potent antimalarial activity in
mouse malaria models. Hence, the present invention comprises the
use of new chemical compounds for the enhanced treatment of
malarial infections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims, and accompanying drawings
wherein:
[0019] FIG. 1 shows the structures of isolated natural product
compounds Formula A and Formula B.
[0020] FIG. 2 shows the structures of newly synthesized derivative
compounds Formula I and Formula II.
[0021] FIG. 3 shows the chemical synthesis route.
DETAILED DESCRIPTION OF THE INVENTION
[0022] As mentioned earlier, the invention provides the following
compounds: Formula I, as shown in FIG. 001, and Formula II, as
shown in FIG. 002.
[0023] The invention is inclusive of the compounds described herein
in any of their pharmaceutically acceptable forms, including
isomers such as diastereomers and enantiomers, salts, solvates,
polymorphs, and the like.
[0024] Isolation of Natural Products Formula A and Formula B.
[0025] General Experimental Procedures. Melting points
(uncorrected) were recorded on an Electrothermal 9100 instrument.
Optical rotations were measured using a JASCO DIP-370 digital
polarimeter in MeOH at ambient temperature. UV spectra were
obtained in MeOH, using a Hewlett-Packard 8452A spectrophotometer.
IR spectra were taken as KBr disks on an Ati Mattson (Genesis
Series) FTIR spectrophotometer. The NMR spectra were recorded on a
Bruker Avance DRX-500 instrument at 500 MHz (.sup.1H) and 125 MHz
(.sup.13C) in appropriate deuteriated solvent. Multiplicity
determinations (DEPT) and 2D NMR spectra (COSY, HMQC, HMBC) were
run using standard Bruker pulse programs. The HRMS were obtained by
direct injection using Bruker Bioapex-FTMS with electrospray
ionization (ESI). TLC was carried out on Silica gel F254 plates,
with appropriate solvent system. For flash column chromatography,
Silica gel from J. T. Baker (40 .mu.m flash) was used. Centrifugal
preparative TLC (using a Chromatotron instrument, Harrison Research
Inc. model 8924) was carried out on 4 mm Silica gel GF Chromatotron
rotors (Analtech, Inc.), with the appropriate solvent system. The
isolated compounds were visualized using UV light, followed by
spraying with anisaldehyde/H.sub.2SO.sub.4 reagent. All solvents
are recycled when possible.
[0026] Extraction and Isolation. The powdered air-dried root barks
of Ocimum sanctum Linn (20 kg) were extracted by percolation with
95% EtOH (35 L.times.3). The combined extracts were evaporated
separately under reduced pressure and then freeze-dried to yield a
thick paste (2.4 kg). A portion of the paste (1.2 kg) was extracted
with 0.05 N hydrochloric acid; the acid solution was extracted with
chloroform. The chloroform was recycled and the residue from the
chloroform solution was discarded. The aqueous phase was then made
basic with sodium carbonate and extracted exhaustively with
chloroform. The combined CHCl.sub.3 fraction was dried over
anhydrous Na.sub.2SO.sub.4 and evaporated under a vacuum to yield
640 g of residue. The CHCl.sub.3 residue (640 g) was flash
chromatographed on silica gel, using CHCl.sub.3--MeOH--NH.sub.4OH
(93.5/6.0/0.5) as eluant to afford a light yellow solid (42 g).
This was crystallized from CHCl.sub.3--MeOH by slow evaporation at
room temperature to give Formula A as off-white needles (35 g).
Further elution with CHCl.sub.3--MeOH--NH.sub.4OH (91.5/8.0/0.5)
followed by concentration yielded a yellow solid (46 g).
Crystallization from CHCl.sub.3--MeOH by slow evaporation at room
temperature gave Formula B as pale yellow needles (39 g). Yield:
Formula A: 0.35% from dried roots; Formula B: 0.39% from dried
roots. The structures of Formula A and Formula B are elucidated by
MS (HRESIMS), IR, UV, and NMR (2D COSY, HMQC, HMBC) methods. Both
compounds possess satisfactory spectroscopic and analytical
data.
[0027] Synthesis of Derivative Compounds Formula I and Formula
II.
[0028] Synthesis of Formula I. Formula A (35 g) was dissolved in
1.0 L of methylene chloride. Pyridine (29 g) was then added in,
followed by the addition of methyl triflate (34 g). The resulting
solution was heated at 80.degree. C. for 2.5 h. It was cooled.
Methylene chloride and pyridine were removed under reduced pressure
in a rotary evaporator. The residue was partitioned between ethyl
acetate (500 ML) and water (500 ML). The organic layer was
separated, washed with brine, and concentrated. Crystallization
from CHCl.sub.3 by slow evaporation at room temperature gave
Formula I as white needles (34 g). Yield: 90%.
[0029] Synthesis of Formula II. Formula B (39 g) was dissolved in
300 ML of 2,6-lutidine. Dimethoxymethane (12 g) was then added in.
The solution was cooled to 0.degree. C. and trimethylsilyl triflate
(23 g) was added in. The resulting solution was vigorously stirred
at 0.degree. C. for 30 min. 2,6-Lutidine was removed under reduced
pressure in a rotary evaporator. The residue was partitioned
between ethyl acetate (500 ML) and water (500 ML). The organic
layer was separated, washed with brine, and concentrated.
Crystallization from CHCl.sub.3 by slow evaporation at room
temperature gave Formula II as white needles (35 g). Yield:
88%.
[0030] The structures of Formula I and Formula II are elucidated by
MS (HRESIMS), IR, UV, and NMR (2D COSY, HMQC, HMBC) methods. Both
compounds possess satisfactory spectroscopic and analytical
data.
##STR00003##
[0031] Formula I (hydrochloride salt): white needles, melting point
182-183.degree. C.; [.alpha.].sub.D+31.5.degree. (c 0.22, MeOH); UV
(MeOH) .lamda..sub.max (log .epsilon.) 233 (4.55), 275 (3.25), 330
(3.40), 360 (3.10) nm; IR (KBr) .nu..sub.max 3030, 2995-2845, 1690,
1665, 1655, 1615, 1580, 1470, 1460, 1370, 1365, 1335, 1285, 1250,
1135, 1080, 865, 830 cm.sup.-1. .sup.1H NMR (CD.sub.3OD, 500 MHz)
.delta..sub.H 8.48 (1H, s, H-8), 8.12 (1H, d, J=6.8 Hz, H-3), 7.33
(1H, d, J=6.8 Hz, H-4), 7.09 (1H, s, H-6), 5.88 (1H, ddd, J=15.4,
8.8 and 5.4 Hz, H-22), 5.21 (1H, dd, J=8.8 and 2.1 Hz, H.sub.a-23),
5.18 (1H, dd, J=15.4 and 2.1 Hz, H.sub.b-23), 4.17 (1H, dd, J=9.2
and 4.4 Hz, H-17), 3.85 (3H, s, H-24), 3.51 (1H, dd, J=7.6 and 5.9
Hz, H-13), 3.44 (1H, d, J=7.6 Hz, H-12), 3.38 (3H, s, H-25), 3.32
(3H, s, H-26), 3.13 (1H, dd, J=5.9 and 5.4 Hz, H-14), 2.55 (1H, dd,
J=16.8 and 9.2 Hz, H.sub.a-16), 2.47 (1H, dd, J=16.8 and 4.4 Hz,
H.sub.b-16), 0.51 (1H, d, J=8.5 Hz, H.sub.a-21), 0.46 (1H, d, J=8.5
Hz, H.sub.b-21). .sup.13C NMR (CD.sub.3OD, 125 MHz) .delta..sub.c
168.4 (C, C-19), 159.9 (C, C-5), 153.8 (C, C-20), 145.1 (C, C-2),
142.3 (C, C-9), 140.8 (CH, C-22), 133.1 (CH, C-3), 131.4 (CH, C-8),
128.9 (C, C-7), 122.4 (CH, C-4), 115.3 (CH.sub.2, C-23), 108.5 (CH,
C-6), 78.8 (CH, C-12), 71.3 (CH, C-13), 58.3 (CH, C-14), 56.9
(CH.sub.3, C-24), 56.0 (CH, C-17), 54.1 (C, C-11), 52.9 (CH.sub.3,
C-25), 52.5 (CH.sub.3, C-26), 48.5 (CH.sub.2, C-16), 33.1 (C,
C-10), 15.2 (CH.sub.2, C-21). HRESIMS m/z 430.1751 [M+Na].sup.+
(calcd for C.sub.23H.sub.25N.sub.3NaO.sub.4.sup.+, 430.1743)
(100%). Anal. Calcd for C.sub.23H.sub.25N.sub.3O.sub.4.HCl: C,
62.23; H, 5.90; Cl, 7.99; N, 9.47. Found: C, 62.31; H, 5.88; Cl,
7.95; N, 9.51.
##STR00004##
[0032] Formula II (hydrochloride salt): white needles, melting
point 184-185.degree. C.; [.alpha.].sub.D-19.2.degree. (c 0.20,
MeOH); UV (MeOH).lamda..sub.max (log .epsilon.) 245 (4.15), 299
(3.35), 340 (3.15), 375 (3.20) nm; IR (KBr) .nu..sub.max, 3035,
2990-2850, 1690, 1675, 1665, 1610, 1590, 1465, 1455, 1375, 1370,
1345, 1280, 1245, 1130, 1085, 860, 835 cm.sup.-1..sup.1H NMR
(CD.sub.3OD, 500 MHz) .delta..sub.H 8.62 (1H, s, H-8), 8.11 (1H, d,
J=6.9 Hz, H-3), 7.39 (1H, d, J=6.9 Hz, H-4), 6.97 (1H, s, H-6),
5.84 (1H, ddd, J=15.6, 8.7 and 5.2 Hz, H-22), 5.25 (1H, dd, J=8.7
and 2.0 Hz, H.sub.a-23), 5.19 (1H, dd, J=15.6 and 2.0 Hz,
H.sub.b-23), 4.99 (2H, s, H-25), 4.23 (1H, dd, J=8.7 and 4.6 Hz,
H-12), 3.88 (3H, s, H-24), 3.55 (1H, dd, J=6.1 and 4.7 Hz, H-16),
3.43 (1H, d, J=4.7 Hz, H-17), 3.21 (1H, dd, J=6.1 and 5.2 Hz,
H-15), 2.58 (1H, dd, J=17.1 and 8.7 Hz, H.sub.a-13), 2.49 (1H, dd,
J=17.1 and 4.6 Hz, H.sub.b-13), 0.53 (1H, d, J=8.6 Hz, H.sub.a-21),
0.48 (1H, d, J=8.6 Hz, H.sub.b-21). .sup.13C NMR (CD.sub.3OD, 125
MHz) .delta..sub.c 168.1 (C, C-10), 165.3 (C, C-20), 158.3 (C,
C-5), 143.1 (C, C-2), 141.8 (CH, C-22), 136.9 (CH, C-8), 130.5 (CH,
C-3), 129.6 (C, C-7), 127.9 (C, C-9), 121.2 (CH, C-4), 115.2
(CH.sub.2, C-23), 107.4 (CH, C-6), 99.8 (CH.sub.2, C-25), 78.6 (CH,
C-17), 70.7 (CH, C-16), 59.1 (CH, C-15), 56.3 (CH.sub.3, C-24),
55.9 (CH, C-12), 54.1 (C, C-18), 48.7 (CH.sub.2, C-13), 32.4 (C,
C-19), 15.2 (CH.sub.2, C-21). HRESIMS m/z 414.1442 [M+Na].sup.+
(calcd for C.sub.22H.sub.21N.sub.3NaO.sub.4.sup.+, 414.1430)
(100%). Anal. Calcd for C.sub.22H.sub.21N.sub.3O.sub.4.HCl: C,
61.75; H, 5.18; Cl, 8.29; N, 9.82. Found: C, 61.82; H, 5.15; Cl,
8.33; N, 9.75.
[0033] It is understood that while a compound of the general
structural formulas herein may exhibit the phenomenon of
tautomerism, the structural formulas within this specification
expressly depict only one of the possible tautomeric forms. It is
therefore to be understood that the structural formulas herein are
intended to represent any tautomeric form of the depicted compound
and is not to be limited merely to a specific compound form
depicted by the structural formulas.
[0034] It is also understood that the structural formulas are
intended to represent any configurational form of the depicted
compound and is not to be limited merely to a specific compound
form depicted by the structural formulas.
[0035] Additionally, the compounds of the invention include
pharmaceutically acceptable salts, multimeric forms, active
metabolites, precursors and salts of such metabolites of the
compounds of the present invention.
[0036] The compound of the present invention is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like, or
with an organic acid, such as acetic acid, maleic acid, succinic
acid, mandelic acid, fumaric acid, malonic acid, pyrvic acid,
oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid,
such as glucuronic acid or galacturonic acid, an .alpha.-hydroxy
acid, such as citric acid or tartaric acid, an amino acid, such as
aspartic acid or glutamic acid, an aromatic acid, such as benzoic
acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic
acid or ethanesulfonic acid, or the like.
[0037] The compound of the present invention are solids, it is
understood by those skilled in the art that the compound of the
present invention and salts may exist in different crystal or
polymorphic forms, all of which are intended to be within the scope
of the present invention and specified structural formulas.
[0038] The compounds of the present invention in accordance with
the present invention are useful in the treatment of malaria and
diseases and disorders associated with malaria or a Plasmodium
parasite.
[0039] Antimalarial Activity
[0040] For in vitro efficacy evaluation, Formula I and Formula II
were tested against two P. falciparum malaria parasite clones: W2
(chloroquine resistant) and D6 (chloroquine sensitive). For in
vitro toxicological studies, the following cell line was chosen:
human adult liver epithelial cells (THLE-3). THLE-3 cells express
phenotypic characteristics of normal adult liver epithelial cells
and constitute a valuable model for pharmacotoxicological studies.
The IC.sub.50 values (50% inhibitory concentrations) were
summarized in Table 1.
TABLE-US-00001 TABLE 1 In Vitro Activity Against Malarial Parasites
and Cell Lines [IC.sub.50 (ng/ml)] Test Compounds D6 Strain W2
Strain THLE-3 Selectivity.sup.a Formula A 13.2 12.9 19800 1500
Formula B 9.7 10.2 19100 1969 Formula I 0.47 0.43 19400 4.1 .times.
10.sup.4 Formula II 0.29 0.33 18900 7.0 .times. 10.sup.4
Chloroquine 6.3 108 2920 463 Mefloquine 3.4 5.6 2700 794
.sup.aSelectivity is calculated as IC.sub.50 (THLE-3)/IC.sub.50
(D6).
[0041] Both natural product Formula A and Formula B possess modest
antimalarial activity. Their corresponding derivatives, Formula I
and Formula II, possess antimalarial activity superior to the
commonly used antimalarial drugs, chloroquine and mefloquine and
have shown potency against both chloroquine sensitive malarial
strain (D-6) and chloroquine resistant malarial strain (W-2).
Noticeably, all new compounds were much less toxic than chloroquine
and mefloquine.
[0042] The blood schizonticidal activity of these new natural
product derivatives Formula I and Formula II was determined as
described herein (Table 2).
TABLE-US-00002 TABLE 2 Antimalarial Activity of Natural Product
Derivatives Formula I and Formula II: Blood schizontocidal activity
against P. berghei in mice: Mice surviving 60 days/Mice infected
and treated. Oral Dose, mg/kg, day; (Total dose, mg/kg) Compound
0.25 (0.75) 1.0 (3.0) 4.0 (12) 16 (48) 64 (192) Chloroquine 0/7 0/7
2/7 4/7 0/7 Formula I 7/7 7/7 7/7 7/7 7/7 Formula II 7/7 7/7 7/7
7/7 7/7 Control 0/7
[0043] Both compounds (Formula I and Formula II) are very active
and much less toxic (no mice showed toxicity at the highest dose
tested). These compounds had also shown excellent activity by the
subcutaneous route of administration [J. Med. Chem., 25, 1094
(1982) M. P. LaMontagne et al.] (See Table 3).
TABLE-US-00003 TABLE 3 Suppressive Antimalarial Activity of New
Natural Product Derivatives Formula I and Formula II: Blood
schizontocidal activity against P. berghei in mice. Subcutaneously
as a single dose. Mice surviving 60 days/Mice infected and treated
Dose, mg/kg Compounds 1 2 4 8 16 32 64 128 Formula I 4/5 5/5 5/5
5/5 5/5 5/5 5/5 5/5 Formula II 4/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5
Control 0/5
[0044] Mice were treated with a single dose of the compound
administered subcutaneously 72 h after infection. Number of cures
is the number of mice surviving, out of five, 60 days
postinfection.
[0045] Compounds were tested for oral prophylactic activity. Mice
treated with a single dose of 0.5 mg/kg of Formula I or Formula II
within the period of 2 days pre-infection through 2 days
post-infection were completely protected from malaria. Data were
summarized in Table 4 and Table 5.
TABLE-US-00004 TABLE 4 Prophylactic Anti-Malarial Activity of
Formula I. Mice infected and treated/Mice surviving day 60 dose,
mg/kg Day of Treatment 0.5 2 8 32 -2 5/5 5/5 5/5 5/5 -1 5/5 5/5 5/5
5/5 1 5/5 5/5 5/5 5/5 2 5/5 5/5 5/5 5/5 Controls 0/5
TABLE-US-00005 TABLE 5 Prophylactic Anti-Malarial Activity of
Formula II. Mice infected and treated/Mice surviving day 60 dose,
mg/kg Day of Treatment 0.5 2 8 32 -2 5/5 5/5 5/5 5/5 -1 5/5 5/5 5/5
5/5 1 5/5 5/5 5/5 5/5 2 5/5 5/5 5/5 5/5 Controls 0/5
[0046] The present invention, hence, relates to the finding that
some new natural product derivative compounds (Formula I and
Formula II) have significantly greater activity and less toxicity
in the treatment of malaria. The present invention relates to new
chemical compositions and also to the use of these compositions as
pharmaceuticals when combined with an acceptable pharmaceutical
carrier in the treatment of malaria.
[0047] Administration of the compounds of the invention may be
parenteral, oral, intravenous, intramuscular, subcutaneous,
intrapleural, intrathecal, intraperitoneal, aerosol or transdermal
administration to achieve the desirable antimalarial effect. These
drugs may be administered as the free base form or in the form of a
pharmaceutically acceptable acid addition salt wherein the acid
addition salt may be either organic or inorganic in nature.
Suitable inorganic acids for salt formation include but are not
restricted to: phosphoric acid, hydrochloric acid or sulfuric acid.
Suitable organic acids for the formation of salts may include but
are not restricted to: succinic acid, citric acid, fumaric acid or
isothionic acid. When administered orally, the compounds of the
invention may be in the form of tablets (single or multilayer,
coated or uncoated) capsules or dragees. These oral formulations
may be admixed with a solid excipient such as lactose, sucrose,
starch, microcrystalline cellulose, magnesium sterate, or talc.
When parenteral administration may be indicated, an aqueous
solution or an oleaginous formulation of the agent may be employed.
Aqueous solutions can be prepared in water or physiological saline,
either with or without buffers. Oleaginous formulation may be made
in natural oils such as peanut oil or olive oil, for example. The
actual dosage amount administered can be determined by physical and
physiological factors such as body weight, severity of condition,
and idiopathy of the subject.
[0048] Biological Testing Procedures
[0049] In Vitro Anti-Malarial Activity Evaluation
[0050] Two P. falciparum malaria parasite clones, W2 and D6, from
Malaria Research and Reference Reagent Resource Center (MR4), were
utilized in susceptibility testing. The W2 clone is susceptible to
mefloquine but resistant to chloroquine, sulfadoxine,
pyrimethamine, and quinine, whereas the D6 clone is naturally
resistant to mefloquine but susceptible to chloroquine,
sulfadoxine, pyrimethamine and quinine. Test compounds were
initially dissolved in DMSO and diluted 400 fold in RPMI 1640
culture medium supplemented with 25 mM Hepes, 32 mM NaHCO.sub.3 and
10% Albumax I (Gibco, N.Y.). These solutions were subsequently
serially diluted two-fold with a Beckman Biomek.RTM. 1000 Robot
(Fullerton, Calif.) over 11 different concentrations. The parasites
were exposed to serial dilutions of each compound for 48 hrs and
incubated at 37.degree. C. with 5% O.sub.2, 5% CO.sub.2 and 90%
N.sub.2 prior to the addition of [.sup.3H]hypoxanthine. After a
further incubation of 18 hrs, parasite DNA was harvested from each
microtiter well using Packard Filtermate.TM. 196 Harvester
(Meriden, Conn.) onto glass filters. Uptake of
[.sup.3H]hypoxanthine was measured with a Packard TopCount Liquid
Scintillation Counter (Packard Instrument Co.).
Concentration-response data was analyzed using a non-linear
regression logistic dose response model and the IC.sub.50 values
(50% inhibitory concentrations) for each compound were
calculated.
[0051] In Vitro Toxicity Evaluation
[0052] Human adult liver epithelial cells (THLE-3) was maintained
in tissue culture flasks in Waymouth's medium (Gibco, N.Y.)
supplemented with 10% fetal bovine serum. Toxicity tests were
performed in 96-well tissue culture plates using the
protein-binding dye Sulforhodamine B. Test compounds were serially
diluted and added to empty wells of the 96-well plate. THLE-3 cells
in their culture medium were immediately seeded into the wells.
Solvent blanks (no compound) were run in each test. After 72 hours
under culture conditions cells were fixed to the plate by layering
50% TCA (4.degree. C.) over the growth media in each well to
produce a final TCA concentration of 10%. After incubating for one
hour at 4.degree. C., cultures were washed five times with tap
water and left air-dried. Wells were stained for 30 min. with 0.4%
(w/v) SRB in 1% acetic acid and washed four times with 1% acetic
acid. Cultures were left air-dried and bound dye was solubilized
with 10 mM Tris base (pH 10.5) for 15 min. on a gyratory shaker at
room temperature. A Spectra MAX Plus Microtiter Plate Reader
(Molecular Devices, Menlo Park, Calif.) was used to measure the
optical density at wavelengths of 490-530 nm. The 50% cell growth
inhibitory concentration (IC.sub.50) value was derived from the
dose-response curve.
[0053] Blood Schizontocidal Test in Animals
[0054] Drugs were mixed in 0.5% hydroxycellulose 0.1% Tween 80 and
administered orally b.i.d. on days 3, 4 and 5 postinfection. CD-1
male or female mice, 5 weeks of age, were infected with
5.times.10.sup.4 parasitized erythrocytes of Plasmodium berghei
KBG-173 mm strain. Blood films were taken on day +6 and weekly
thereafter until day +60. Parasitemias were calculated and SD90
value (dose suppressing 90% of the parasites in treated groups
compared with the infected non-treated controls) on day +6
postinfection. Mortality data was tabulated for 60 days at which
time all mice surviving that were blood film negative were
considered cured.
[0055] Compounds were tested at three dose levels, 4, 1, and 0.25
mg/kg body weight per day. The activity of these compounds were
compared with the untreated control: In untreated controls, death
occurs within 8-9 days. Compounds which are effective against
Plasmodium berghei infection increase the mean survival time of the
infected animals when compared with the untreated controls. Mice
that survive after thirty days and are free of parasites in blood
are considered cured.
[0056] Efficacy of the drug is determined by the number of cures at
the end of a 30 day period and the increase in mean survival time
over the control (.DELTA.MST). The effect of the test drugs also
could be determined by the reduction of the parasitemia (percentage
of the red blood cells detected with the parasites) over the
untreated control on day 6, one day after the treatment is
completed. Both these methods yield virtually identical results. If
the dose of test compounds are inadequate, after initial clearance,
residual parasites will multiply and relapses will occur within
thirty days.
[0057] Prophylactic Test in Animals
[0058] Drugs were mixed in 0.5% hydroxycellulose 0.1% Tween 80 and
administered orally b.i.d. either on day 5, 4, 3, 2, or 1 prior to
the infection or 1 or 2 days postinfection. CD-I male or female
mice, 5 weeks of age, were infected with 5.times.10.sup.4
parasitized erythrocytes of Plasmodium berghei KBG-173 mm strain.
Blood films were taken on day +6 and weekly thereafter until
day+30. Mortality data was tabulated for 30 days at which time all
mice surviving that were blood film negative were considered
cured.
[0059] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
can be used in the practice of testing of the present invention,
the preferred methods and materials are described herein.
[0060] It is understood that the examples and embodiments described
herein are for illustrative purposes only and the various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
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